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Detection

المؤلف:  Sue Jickells , Adam Negrusz (Editors)

المصدر:  Clarkes Analytical Forensic Toxicology

الجزء والصفحة:  p355-356

2026-11-02

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Detection

About 1–10 lg of substances derivatised to form a coloured product can be detected by visual inspection of a TLC plate. The quantitative reproducibility is rarely better than 10–30%. This may be adequate for qualitative methods, but for reliable quantification, in-situ spectrophotometric methods are preferred as they are more accurate and far less tedious and time consuming than excising zones from the layer for determination by conventional solution spectrophotometry. A fluorescence-quenching technique can be used for detection. This involves visualisation of UV-absorbing compounds on TLC plates that incorporate a fluorescent indicator. The zones of UV-absorbing substance appear dark against the brightly fluorescing background of a lighter colour when the plate is exposed to UV light of short wavelength. The method is not universal, since it requires overlap between the absorption bands of the indicator (kmax ~ 280 nm with virtually no absorption below 240 nm) and the drug, but in favourable cases it is a valuable and nondestructive method for zone location.

All optical methods for the quantitative in-situ evaluation of TLC chromatograms are based upon measuring the difference in optical response between a sample-free region of the layer and regions of the layer in which separated substances are present. Reflectance measure ments can be made at any wavelength from the UV to the near infrared (185 to 2500 nm). The relationship between signal and sample amount in the absorption mode is nonlinear, and does not conform to any simple equation. The principal method of quantification in TLC is by calibration using a series of standards that span the concentration range of the drug to be determined. The calibration curve is usually based on a second-order polynomial fit for the calibration standards, with individual samples quantified by interpolation only.

The determination of drugs that fluoresce on TLC plates is fundamentally different from absorption measurements. At low sample concentrations the fluorescence signal F is described adequately by F=QI₀ εbC, where u is the quantum yield, I0 is the intensity of the excitation source, e is the molar absorption coefficient, b is the thickness of the TLC layer and C is the sample amount. With the exception of the sample amount, all terms in this expression are constant, or fixed by the experiment, and therefore the fluorescence emission is linearly dependent on the sample amount (over two or three orders of magnitude).